Multi-modal cell-free DNA genomic and fragmentomic patterns enhance cancer survival and recurrence analysis.

The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.

Acidosis-mediated increase in IFN-γ-induced PD-L1 expression on cancer cells as an immune escape mechanism in solid tumors.

Immune checkpoint inhibitors have revolutionized cancer therapy, yet the efficacy of these treatments is often limited by the heterogeneous and hypoxic tumor microenvironment (TME) of solid tumors. In the TME, programmed death-ligand 1 (PD-L1) expression on cancer cells is mainly regulated by Interferon-gamma (IFN-γ), which induces T cell exhaustion and enables tumor immune evasion. In this study, we demonstrate that acidosis, a common characteristic of solid tumors, significantly increases IFN-γ-induced PD-L1 expression on aggressive cancer cells, thus promoting immune escape. Using preclinical models, we found that acidosis enhances the genomic expression and phosphorylation of signal transducer and activator of transcription 1 (STAT1), and the translation of STAT1 mRNA by eukaryotic initiation factor 4F (elF4F), resulting in an increased PD-L1 expression. We observed this effect in murine and human anti-PD-L1-responsive tumor cell lines, but not in anti-PD-L1-nonresponsive tumor cell lines. In vivo studies fully validated our in vitro findings and revealed that neutralizing the acidic extracellular tumor pH by sodium bicarbonate treatment suppresses IFN-γ-induced PD-L1 expression and promotes immune cell infiltration in responsive tumors and thus reduces tumor growth. However, this effect was not observed in anti-PD-L1-nonresponsive tumors. In vivo experiments in tumor-bearing IFN-γ-/- mice validated the dependency on immune cell-derived IFN-γ for acidosis-mediated cancer cell PD-L1 induction and tumor immune escape. Thus, acidosis and IFN-γ-induced elevation of PD-L1 expression on cancer cells represent a previously unknown immune escape mechanism that may serve as a novel biomarker for anti-PD-L1/PD-1 treatment response. These findings have important implications for the development of new strategies to enhance the efficacy of immunotherapy in cancer patients.

Real-time analysis of the cancer genome and fragmentome from plasma and urine cell-free DNA using nanopore sequencing.

Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.

Predictive Value of Combined Positive Score and Tumor Proportion Score for Immunotherapy Response in Advanced NSCLC.

Introduction: In advanced-stage NSCLC, tumor proportion score (TPS) is typically used to predict the efficacy of immune checkpoint inhibitors (ICIs). Nevertheless, in other cancer types, the combined positive score (CPS), which covers programmed death-ligand 1 (PD-L1) expression on both tumor and surrounding immune cells, is used. We investigated the predictive value of CPS in comparison to TPS in advanced NSCLC. Methods: A monocenter, retrospective study was performed in patients with advanced NSCLC treated with ICI monotherapy between 2015 and 2021. Hematoxylin and eosin and PD-L1 were stained on baseline tumor biopsy samples to score PD-L1 by both TPS and CPS. Positivity for TPS and CPS was defined as a score of 1% or above. Progression-free survival and overall survival (OS) were assessed for TPS and CPS scores. Results: Among the 187 included patients, PD-L1 positivity was found in 112 patients (59.9%) by TPS and 135 patients (72.2%) by CPS. There was no significant difference in OS between TPS- and TPS+ patients (p = 0.20). Nevertheless, CPS+ patients did have a longer OS than CPS- patients (p = 0.006). OS was superior in both TPS-/CPS+ and TPS+/CPS+ as compared with TPS-/CPS- cases (p = 0.018 and p = 0.015, respectively), whereas no considerable differences in OS were found between TPS-/CPS+ and TPS+/CPS+ cases. Conclusions: This retrospective real-world population study revealed that CPS differentiated OS better than TPS in patients with advanced NSCLC with ICI monotherapy. Remarkably, this was driven by the performance of the TPS-/CPS+ subgroup, indicating that CPS may be a better predictive biomarker for ICI efficacy.

A Bispecific γδ T-cell Engager Targeting EGFR Activates a Potent Vγ9Vδ2 T cell-Mediated Immune Response against EGFR-Expressing Tumors.

Vγ9Vδ2 T cells are effector cells with proven antitumor efficacy against a broad range of cancers. This study aimed to assess the antitumor activity and safety of a bispecific antibody directing Vγ9Vδ2 T cells to EGFR-expressing tumors. An EGFR-Vδ2 bispecific T-cell engager (bsTCE) was generated, and its capacity to activate Vγ9Vδ2 T cells and trigger antitumor activity was tested in multiple in vitro, in vivo, and ex vivo models. Studies to explore safety were conducted using cross-reactive surrogate engagers in nonhuman primates (NHP). We found that Vγ9Vδ2 T cells from peripheral blood and tumor specimens of patients with EGFR+ cancers had a distinct immune checkpoint expression profile characterized by low levels of PD-1, LAG-3, and TIM-3. Vγ9Vδ2 T cells could be activated by EGFR-Vδ2 bsTCEs to mediate lysis of various EGFR+ patient-derived tumor samples, and substantial tumor growth inhibition and improved survival were observed in in vivo xenograft mouse models using peripheral blood mononuclear cells (PBMC) as effector cells. EGFR-Vδ2 bsTCEs exerted preferential activity toward EGFR+ tumor cells and induced downstream activation of CD4+ and CD8+ T cells and natural killer (NK) cells without concomitant activation of suppressive regulatory T cells observed with EGFR-CD3 bsTCEs. Administration of fully cross-reactive and half-life extended surrogate engagers to NHPs did not trigger signals in the safety parameters that were assessed. Considering the effector and immune-activating properties of Vγ9Vδ2 T cells, the preclinical efficacy data and acceptable safety profile reported here provide a solid basis for testing EGFR-Vδ2 bsTCEs in patients with EGFR+ malignancies.

Preclinical Identification Of Tumor-Draining Lymph Nodes Using a Multimodal Non-invasive In vivo Imaging Approach.

PURPOSE: Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification. PROCEDURES: For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol-1) and IRDye® 800CW polyethylene glycol (PEG; 25,000-60,000 g mol-1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol-1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis. RESULTS: The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo 18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo 18F-FDG PET/MRI results. CONCLUSIONS: When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN.

Immunological Responses to Cancer Therapy.

The use of immunotherapy for cancer has taken flight in the last two decades, from experimental therapy envisioned mainly by laboratory scientists to everyday treatment used by physicians to treat many patients [...].

Local delivery of low-dose anti-CTLA-4 to the melanoma lymphatic basin leads to systemic Treg reduction and effector T cell activation.

Preclinical studies show that locoregional CTLA-4 blockade is equally effective in inducing tumor eradication as systemic delivery, without the added risk of immune-related side effects. This efficacy is related to access of the CTLA-4 blocking antibodies to tumor-draining lymph nodes (TDLNs). Local delivery of anti-CTLA-4 after surgical removal of primary melanoma, before sentinel lymph node biopsy (SLNB), provides a unique setting to clinically assess the role of TDLN in the biological efficacy of locoregional CTLA-4 blockade. Here, we have evaluated the safety, tolerability, and immunomodulatory effects in the SLN and peripheral blood of a single dose of tremelimumab [a fully human immunoglobulin gamma-2 (IgG2) mAb directed against CTLA-4] in a dose range of 2 to 20 mg, injected intradermally at the tumor excision site 1 week before SLNB in 13 patients with early-stage melanoma (phase 1 trial; NCT04274816). Intradermal delivery was safe and well tolerated and induced activation of migratory dendritic cell (DC) subsets in the SLN. It also induced profound and durable decreases in regulatory T cell (Treg) frequencies and activation of effector T cells in both SLN and peripheral blood. Moreover, systemic T cell responses against NY-ESO-1 or MART-1 were primed or boosted (N = 7), in association with T cell activation and central memory T cell differentiation. These findings indicate that local administration of anti-CTLA-4 may offer a safe and promising adjuvant treatment strategy for patients with early-stage melanoma. Moreover, our data demonstrate a central role for TDLN in the biological efficacy of CTLA-4 blockade and support TDLN-targeted delivery methods.

Comparison of the tumor microenvironments of squamous cell carcinoma at different anatomical locations within the upper aerodigestive tract in relation to response to ICI therapy.

Immunotherapy with immune checkpoint inhibitors (ICI) has improved treatment outcomes in many cancer types and has focused attention on cancer immunity and the role of the tumor microenvironment (TME). Studies into efficacy of immunotherapy and TME are generally restricted to tumors in one anatomical location, while the histological type may have substantial influence on the contexture of the TME, perhaps more so than anatomical location, and subsequently to the response to immunotherapy. This review aims to focus on the TME in ICI-treated tumors of the same histological type, namely carcinogen-induced squamous cell carcinoma developing within the aerodigestive tract, at three locations, i.e. head and neck (HNSCC), esophagus (ESCC) and lung (LUSC).

Targeting Endoglin Expressing Cells in the Tumor Microenvironment Does Not Inhibit Tumor Growth in a Pancreatic Cancer Mouse Model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and is known to have low immunogenicity and an immunosuppressive microenvironment. It is also characterized by high accumulation of dense stroma, composed of mostly cancer-associated fibroblasts (CAFs). Multiple subsets of CAFs are described, with one of them expressing the transforming growth factor (TGF)-ß co-receptor endoglin. In previous work, we and others have shown that endoglin-expressing CAFs stimulate tumor progression and metastasis. Therefore, in this study, we set out to investigate the role of endoglin-expressing CAFs in pancreatic cancer progression. METHODS: First, we investigated the expression of endoglin on CAFs in both human tissues as well as a mouse model for PDAC. Since CAF-specific endoglin expression was high, we targeted endoglin by using the endoglin neutralizing antibody TRC105 in the murine KPC model for PDAC. RESULTS: Although some signs of immune activation were observed, TRC105 did not affect tumor growth. Since 90% of the CD8+ T-cells expressed the immune checkpoint PD-1, we investigated the combination with a PD1 checkpoint inhibitor, which did not enhance therapeutic responses. Finally, genetic deletion of endoglin from collagen 1a1 expressing cells also did not affect the growth of the mouse KPC tumors. CONCLUSION: Our results show that although endoglin is highly expressed on PDAC-CAFs and signaling is efficiently inhibited by TRC105, this does not result in decreased tumor growth in the KPC model for pancreatic cancer.

Immune Checkpoint Therapy: Tumor Draining Lymph Nodes in the Spotlights.

Tumor-draining lymph nodes play a paradoxical role in cancer. Surgeons often resect these sentinel lymph nodes to determine metastatic spread, thereby enabling prognosis and treatment. However, lymph nodes are vital organs for the orchestration of immune responses, due to the close encounters of dedicated immune cells. In view of the success of immunotherapy, the removal of tumor-draining lymph nodes needs to be re-evaluated and viewed in a different light. Recently, an important role for tumor-draining lymph nodes has been proposed in the immunotherapy of cancer. This new insight can change the use of immune checkpoint therapy, particularly with respect to the use in neoadjuvant settings in which lymph nodes are still operational.

Immunotherapy Goes Local: The Central Role of Lymph Nodes in Driving Tumor Infiltration and Efficacy.

Immune checkpoint blockade (ICB) has changed the therapeutic landscape of oncology but its impact is limited by primary or secondary resistance. ICB resistance has been related to a lack of T cells infiltrating into the tumor. Strategies to overcome this hurdle have so far focused on the tumor microenvironment, but have mostly overlooked the role of tumor-draining lymph nodes (TDLN). Whereas for CTLA-4 blockade TDLN have long since been implicated due to its perceived mechanism-of-action involving T cell priming, only recently has evidence been emerging showing TDLN to be vital for the efficacy of PD-1 blockade as well. TDLN are targeted by developing tumors to create an immune suppressed pre-metastatic niche which can lead to priming of dysfunctional antitumor T cells. In this review, we will discuss the evidence that therapeutic targeting of TDLN may ensure sufficient antitumor T cell activation and subsequent tumor infiltration to facilitate effective ICB. Indeed, waves of tumor-specific, proliferating stem cell-like, or progenitor exhausted T cells, either newly primed or reinvigorated in TDLN, are vital for PD-1 blockade efficacy. Both tumor-derived migratory dendritic cell (DC) subsets and DC subsets residing in TDLN, and an interplay between them, have been implicated in the induction of these T cells, their imprinting for homing and subsequent tumor control. We propose that therapeutic approaches, involving local delivery of immune modulatory agents for optimal access to TDLN, aimed at overcoming hampered DC activation, will enable ICB by promoting T cell recruitment to the tumor, both in early and in advanced stages of cancer.

Targeting Endoglin-Expressing Regulatory T Cells in the Tumor Microenvironment Enhances the Effect of PD1 Checkpoint Inhibitor Immunotherapy.

PURPOSE: Endoglin is a coreceptor for TGFß ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody. EXPERIMENTAL DESIGN: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor-knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg). RESULTS: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens. CONCLUSIONS: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

Thermosensitive hydrogels as sustained drug delivery system for CTLA-4 checkpoint blocking antibodies.

Thermosensitive poloxamer 407 (P407) hydrogels were evaluated as slow release system for optimizing CTLA-4 therapy. Slow release reduces systemic antibody levels and potentially mitigates the side effects of CTLA-4 therapy. The 25% P407 hydrogel is injectable at room temperature and depots are established quickly after subcutaneous injection. Scanning electron microscopy revealed the porous structure of the hydrogel, average pore surface was 1335 µm2. Release studies were optimized using the human IgG antibody. IgG was easily incorporated in the hydrogel by simple mixing and no antibodies were lost during preparation. In vitro, hydrogels showed low burst release within the first 24 h. Total IgG load was gradually released within 120 h. In vitro cytotoxicity assays showed that P407 is not cytotoxic and induces no immune activation by itself. In vivo, P407 hydrogels significantly reduced serum IgG levels, were biocompatible and were broken down 1 week after injection. Finally, local hydrogel delivery of anti-CTLA-4 antibodies near established tumors effectively slowed down tumor growth, whilst significantly reduced serum anti-CTLA-4 levels. Altogether, P407 hydrogels represent promising delivery systems for the optimization of CTLA-4 blocking therapy.

Immunogenicity of rat-neu+ mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment.

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours.

Dendritic cells dictate responses to PD-L1 blockade cancer immunotherapy.

PD-L1/PD-1 blocking antibodies have demonstrated therapeutic efficacy across a range of human cancers. Extending this benefit to a greater number of patients, however, will require a better understanding of how these therapies instigate anticancer immunity. Although the PD-L1/PD-1 axis is typically associated with T cell function, we demonstrate here that dendritic cells (DCs) are an important target of PD-L1 blocking antibody. PD-L1 binds two receptors, PD-1 and B7.1 (CD80). PD-L1 is expressed much more abundantly than B7.1 on peripheral and tumor-associated DCs in patients with cancer. Blocking PD-L1 on DCs relieves B7.1 sequestration in cis by PD-L1, which allows the B7.1/CD28 interaction to enhance T cell priming. In line with this, in patients with renal cell carcinoma or non-small cell lung cancer treated with atezolizumab (PD-L1 blockade), a DC gene signature is strongly associated with improved overall survival. These data suggest that PD-L1 blockade reinvigorates DC function to generate potent anticancer T cell immunity.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

Tumor-draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy.

PD-1/PD-L1 checkpoint therapy for cancer is commonly considered to act by reactivating T cells in the tumor microenvironment. Here, we present data from 2 mouse tumor models demonstrating an essential involvement of tumor-draining lymph nodes in PD-1 and PD-L1 therapeutic efficacy. Immune activation induced by checkpoint treatment was predominantly observed in the tumor-draining, but not nondraining, lymph nodes and was reflected in local accumulation of CD8+ T cells. Surgical resection of these lymph nodes, but not contralateral lymph nodes, abolished therapy-induced tumor regressions and was associated with decreased immune infiltrate in the tumor microenvironment. Moreover, inhibitor FTY720, which locks lymphocytes in lymph organs, also abrogated checkpoint therapy, suggesting that the tumor-draining lymph nodes function as sites of T cell invigoration required for checkpoint blockade therapy. Now that PD-1/PD-L1 checkpoint treatment is applied in earlier clinical stages of cancer, our preclinical data advocate for enrolling patients with their tumor-draining lymph nodes still in place, to optimally engage the antitumor immune response and thereby enhance clinical benefit.

High FcγR Expression on Intratumoral Macrophages Enhances Tumor-Targeting Antibody Therapy.

Therapy with tumor-specific Abs is common in the clinic but has limited success against solid malignancies. We aimed at improving the efficacy of this therapy by combining a tumor-specific Ab with immune-activating compounds. In this study, we demonstrate in the aggressive B16F10 mouse melanoma model that concomitant application of the anti-TRP1 Ab (clone TA99) with TLR3-7/8 or -9 ligands, and IL-2 strongly enhanced tumor control in a therapeutic setting. Depletion of NK cells, macrophages, or CD8+ T cells all mitigated the therapeutic response, showing a coordinated immune rejection by innate and adaptive immune cells. FcγRs were essential for the therapeutic effect, with a dominant role for FcγRI and a minor role for FcγRIII and FcγRIV. FcγR expression on NK cells and granulocytes was dispensable, indicating that other tumoricidal functions of NK cells were involved and implicating that FcγRI, -III, and -IV exerted their activity on macrophages. Indeed, F4/80+Ly-6C+ inflammatory macrophages in the tumor microenvironment displayed high levels of these receptors. Whereas administration of the anti-TRP1 Ab alone reduced the frequency of these macrophages, the combination with a TLR agonist retained these cells in the tumor microenvironment. Thus, the addition of innate stimulatory compounds, such as TLR ligands, to tumor-specific Ab therapy could greatly enhance its efficacy in solid cancers via optimal exploitation of FcγRs.